SCREENING METHOD OF APTAMER AND IMMUNOASSAY USING THE APTAMER

Abstract

The present invention relates to an aptamer screening method, and the aptamer screened by the screening method of the present invention binds to a site other than a site where the antibody binds to the target substance to be applicable in various fields such as sandwich-type biosensors and reduce a significant time without requiring a separate pairing selection process. In addition, such an aptamer has excellent stability and sensitivity compared to conventional preparations comprising an antibody, can be mass-produced at low cost in a short time by a chemical synthesis method, and is easily transformed in various ways to increase a binding force. In addition, the immunoassay method using the aptamer screened by the aptamer screening method of the present invention selectively amplifies only the aptamer binding to the target substance in a heterogeneous sandwich structure to detect a relative fluorescence signal, thereby detecting the target substance sensitively and quickly.

Claims

1. A screening method of an aptamer comprising: an antibody immobilization step of immobilizing a specific antibody to a target substance to a support; a first reaction step of adding and reacting the target substance to the antibody-immobilized support; a second reaction step of adding and reacting an aptamer library to the support where the first reaction step is completed; and a first elution step of eluting the aptamer binding to the target substance in the second reaction step.

2. The screening method of the aptamer of claim 1, further comprising: an amplification step of amplifying a nucleic acid of the aptamer eluted in the first elution step; a second-1 reaction step of adding and reacting the amplified nucleic acid of the aptamer into the support on which the first reaction step has been completed; and a second elution step of eluting the aptamer binding to the target substance in the second-1 reaction step.

3. The screening method of the aptamer of claim 2, wherein the amplification step is performed through a polymerase chain reaction.

4. The screening method of the aptamer of claim 2, wherein the amplification step; the second-1 reaction step; and the second elution step may be repeated 2 to 30 times in sequence.

5. The screening method of the aptamer of claim 1, further comprising: after the first reaction step, a step of adding and reacting the aptamer library to the support immobilized with the antibody; and a recovery step of recovering a supernatant containing the aptamer library that has not reacted with the antibody.

6. The screening method of the aptamer of claim 1, wherein the aptamer library includes at least one single-stranded nucleotide sequence selected from the group consisting of different nucleotide sequences.

7. The screening method of the aptamer of claim 6, wherein the single-stranded nucleotide sequence consists of a forward or reverse primer nucleotide sequence for amplification at both terminals, and the center of the primer nucleotide sequence consists of 30 to 50 nucleotide sequences.

8. The screening method of the aptamer of claim 1, wherein the target substance is at least one selected from the group consisting of metal ions, compounds, nucleic acids, proteins, peptides and cells.

9. An aptamer screened by the screening method of claim 1.

10. An immunoassay method comprising: mixing a detection sample with an aptamer of claim 9 specifically binding to a target substance to be detected; reacting the mixture with a binding substance immobilized to the support and specifically binding to the target substance to form a complex of aptamer-target substance-binding substance; and amplifying the aptamer.

11. The immunoassay method of claim 10, further comprising: removing an aptamer that has not formed the complex after the step of forming the complex.

12. The immunoassay method of claim 10, further comprising: adding an amplification reagent before the step of amplifying the aptamer.

13. The immunoassay method of claim 10, further comprising: measuring fluorescence after amplifying the aptamer.

14. The immunoassay method of claim 10, wherein the binding substance is at least one selected from the group consisting of antibodies, antigens, nucleic acids, aptamers, hapten, antigen proteins, DNA, RNA binding proteins, and cationic polymers.

15. The immunoassay method of claim 12, wherein the amplification reagent includes a primer, dNTP, a reaction buffer, a recombinase, and an intercalating dye inserted into the amplified dsDNA to indicate a fluorescence signal.

16. The immunoassay method of claim 10, wherein the amplifying of the aptamer is performed by any one isothermal amplification method selected from the group consisting of helicase-dependent amplification (HAD), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), loop mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), signal mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), isothermal multiple displacement amplification (IMDA), single primer isothermal amplification (SPIA) and circular helicase dependent amplification (cHDA).

17. The immunoassay method of claim 10, wherein the concentration of the binding substance is 0.1 to 100 ng/ml.

18. The immunoassay method of claim 10, wherein the concentration of the aptamer is 0.01 to 10 pM.

19. The immunoassay method of claim 10, wherein the amplifying of the aptamer is performed for 8 minutes to 30 minutes.

20. The immunoassay method of claim 10, wherein a low limit of detection (LOD) of the method is that the concentration of the target substance contained in the sample is 1 fg/mL or more.

Description

DESCRIPTION OF DRAWINGS

[0108] FIG. 1 illustrates a result of confirming the size of a purified nuclear protein of severe fever with thrombocytopenia syndrome (SFTS) virus through SDS-PAGE according to an embodiment of the present invention.

[0109] FIG. 2 illustrates a schematic diagram of each step of a modified SELEX method according to an embodiment of the present invention.

[0110] FIGS. 3A to 3C are graphs showing a result of confirming a target binding force of an aptamer screened according to a modified SELEX method according to an embodiment of the present invention.

[0111] FIG. 4 illustrates a result of confirming binding specificity to nuclear proteins of SFTS virus according to an embodiment of the present invention.

[0112] FIGS. 5A and 5B illustrate a result of confirming a binding force of an aptamer according to a nuclear protein concentration of SFTS virus according to an embodiment of the present invention.

[0113] FIG. 6 illustrates schematically a principle of an immunoassay method (H-sandwich RPA) using an aptamer screened by an aptamer screening method of the present invention, wherein a sample containing an antigen and an aptamer is pre-incubated in a well immobilized with a capture antibody. The aptamer binding to the antigen is amplified during an RPA reaction and detected by an intercalating dye.

[0114] FIGS. 7A to 7D are graphs showing optimization results of factors affecting an immunoassay method (H-sandwich RPA) using an screened aptamer, wherein FIG. 7A illustrates a concentration of a coating antibody used for preparing an immobilized well, FIG. 7B illustrates a concentration of an aptamer reacting with an antigen, FIG. 7C illustrates a concentration of an intercalating dye, and FIG. 7D illustrates relative fluorescent strength saturation for an RPA reaction, and concentrations of an influenza A NP and an aptamer were set at 0.5 pg/mL and 1 μM, respectively.

[0115] FIGS. 8A and 8B illustrate intercalating dye efficiency under various amplification conditions, wherein FIG. 8A illustrates relative fluorescence intensity through a amplification reaction (a: conventional PCR (30 cycles), b: RPA, c: H-sandwich RPA (NP and aptamer are 0.5 pg/mL and 1 pM, respectively), and FIG. 8B illustrates a gel shift image under each amplification reaction (OFF: no aptamer or NP, ON: aptamer 1 pM, NP).

[0116] FIGS. 9A to 9E illustrate results of measuring target concentrations using an immunoassay method (H-sandwich RPA) using a screened aptamer, wherein FIG. 9A illustrates influenza A NP, FIG. 9B illustrates influenza B NP, FIG. 9C illustrates HIV-1 p24, FIG. 9D illustrates Ebola NP, and FIG. 9E illustrates SARS-Cov-2 NP. Each antigen is in a concentration range of 0.001 to 10 pg/mL.

[0117] FIGS. 10A and 10B are graphs showing specificity of H-sandwich RPA based on target-aptamer specificity, wherein FIG. 10A illustrates cross-reactivity between an influenza A NP and an influenza B NP (Blue bar: influenza A NP-specific aptamer 1 pM, influenza A and B NPs were applied at 1 and 10 pg/mL, respectively, Green bar: influenza B NP-specific aptamer 1 pM, influenza A and B NPs were applied at 10 and 1 pg/mL, respectively), and FIG. 10B illustrates cross-reactivity of p24 and Ebola NP (Orange bars: p24-specific aptamer 1 pM, p24 and Ebola NPs were applied at 1 and 10 pg/mL, respectively, Pink bar: Ebola NP-specific aptamer 1 pM, p24 and Ebola NP are applied at 10 and 1 pg/mL, respectively).

MODES FOR THE INVENTION

[0118] Hereinafter, the present invention will be described in more detail with reference to Examples. These Examples are to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these Examples in accordance with the gist of the present invention.

<Example 1> Screening Method of Aptamer

[0119] 1. Design of Single Strand DNA Aptamer (ssDNA) Library

[0120] In an aptamer screening method, in an initial round, an ssDNA oligonucleotide library randomly containing 40 nucleotide sequences (SEQ ID NO: 2: 5′-ATC CAG AGT GAC GCA GCA-[core sequence; (N X 40)]-TG GAC ACG GTG GCT TAG T-3′) was used. All oligonucleotides used in this study were obtained from Integrated DNA Technologies Inc. (Coralville, Iowa, USA). At this time, the ssDNA library was put in a binding buffer (50 mM Tris-hydrochloric acid, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2), denatured by heating at 90° C. for 5 minutes, and then washed with 0.01% Tween 20, and immediately cooled on ice for 10 minutes, and then used.

[0121] 2. Expression and Purification of Nuclear Protein of Server Fever with Thrombocytopenia Syndrome (SFTS) Virus

[0122] A PCR product was obtained by amplifying a nucleotide sequence encoding a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 through a polymerase chain reaction (PCR). Thereafter, the PCR product was inserted into a pET28a expression vector and then transformed to E. coli. BL21 and shake-incubated at 37° C. When an OD.sub.600 value of a shake-culture medium reached 0.6, 1 mM isoprophyl-b-D-thiogalactopyranoside (IPTG) was treated and incubated overnight at 25° C. to induce protein expression. Thereafter, the transformant was applied with ultrasonic waves to destroy cells, and then a protein represented by SEQ ID NO: 1 was obtained from the cell lysate.

[0123] As illustrated in FIG. 1, as a result of performing SDS-PAGE on the isolated protein according to a general method, it was confirmed that a nuclear protein of server fever with thrombocytopenia syndrome (hereinafter referred to as ‘SFTS’) virus, as a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 was obtained.

[0124] 3. Screening Method of Aptamer

[0125] (1) Each Step of Screening Method of Aptamer

[0126] As illustrated in FIG. 2, the screening method of the aptamer of the present invention was performed to screen an aptamer capable of specifically binding to the SFTS virus. Specifically, the screening method of the modified aptamer was as follows.

[0127] 1) Antibody Immobilizing and Blocking Steps

[0128] Antibody immobilizing step: The antibody specifically binding to the amino acid sequence represented by SEQ ID NO: 1 was diluted in a 0.1 M carbonate buffer in pH 9.5, put in a 96-well plate and then reacted overnight at 4° C. to obtain the antibody on the 96-well plate.

[0129] Blocking step: Thereafter, 200 μl of a washing buffer (potassium phosphate buffer (PBS) containing 0.01% Tween) was added and washed in the coated plate. Non-specific binding reaction that may occur when ssDNA bound to the antibody was blocked by adding 1% bovine serum albumin (BSA) in the washed plate and reacting for 1 hour at room temperature.

[0130] 1-1) Recovery Step:

[0131] In step 1), in order to reduce non-specific binding possibility between a protein except for the amino acid sequence represented by SEQ ID NO: 1 and ssDNA, a recovery step was additionally performed, in which a buffer including the ssDNA library was added to the blocked plate using the BSA and incubated for 30 minutes, and then a supernatant (hereinafter, referred to as a ‘supernatant’) containing only the ssDNA library without binding to the antibody was obtained.

[0132] 2) First Reaction Step; Antigen-Antibody Reaction

[0133] The protein of Example 1 diluted in PBS at a concentration of 1 μg/mL was added to the plate on which the antibody immobilization and blocking were completed in step 1), and incubated at room temperature for 2 hours.

[0134] 3) Second Reaction Step; Step of Binding to ssDNA Library

[0135] The supernatant of 1-1) was added to the plate on which the first reaction step was completed, reacted at room temperature for 1 hour, and then washed 5 times using 200 μl of a wash buffer.

[0136] 4) First Elution Step; ssDNA Elution and Amplification Step

[0137] A binding buffer was added to the plate washed in step 2), and reacted at 80° C. for 10 minutes to elute ssDNA bound to the protein of Example 1.

[0138] 5) Amplification Step; Third Reaction Step; and Second Elution Step

[0139] The ssDNA eluate of the first elution step 4) was purified according to a method provided by a manufacturer using a PCR purification kit, and then the purified ssDNA was dissolved in 30 μl of sterile water. The purified ssDNA was mixed with 1 μM of a primer pair, a 2.5 mM dNTP mixture, and 1.2 U of Pfu polymerase to make a final volume of 50 μl, and then polymerase chain reaction was performed to obtain amplified ssDNA. At this time, in the case of the polymerase chain reaction, 25 cycles were performed under conditions of 5 min at 95° C.; 30 sec at 95° C.; 20 sec at 57° C.; and 20 sec at 72° C. and the final extension was performed under a condition of 5 min at 72° C.

[0140] Then, the amplified ssDNA was subjected to a third reaction step in the same manner as in the ‘3) second reaction step’, and subjected to a second elution step in the same manner as in the ‘4) first elution step’, and the amplification step; the third reaction step and the second elution step were performed at least 10 times. Through this process, three ssDNA sequences were finally screened.

[0141] (2) ssDNA Sequencing

[0142] The three types of ssDNA sequences selected by the aptamer screening method were amplified through the same polymerase chain reaction as in the aptamer screening method, and the amplified products were inserted into the pETHis6 TEVLIC cloning vector (Addgene, USA), respectively. Then, after transforming the vector into E. Coli DH5α, the amplified plasmid was isolated from the transformant to perform DNA sequencing. Here, the DNA sequencing was performed in Macrogen (Korea), and the secondary structure of the aptamer as ssDNA was predicted using an Mfold program based on a Zuker algorithm.

[0143] As a result, the screened aptamer had a nucleotide sequence of 5′-ATC CAG AGT GAC GCA GCA-[core sequence; (N X 40)]-TG GAC ACG GTG GCT TAG T-3′ of SEQ ID NO: 2 and the nucleotide sequence was the same as described in Table 1 below.

TABLE-US-00002 TABLE 1 SEQ ID Name NO: Core sequence Aptamer 1 SEQ ID CGACCACAGATTGG NO: 3 AGACTGATAGTGCA CGAGCAAGGACA Aptamer 2 SEQ ID TCGGATGGATTGTG NO: 4 GTCGAAGTTGTTTC CGACACTAGTCA Aptamer 3 SEQ ID CACATCGGAGAACA NO: 5 GGCGCACTGTCGG AGGAACCGCAACG

[0144] A phosphor (FAM) (in the case of FAM-labeled aptamers, hereinafter referred to as ‘FAM-aptamer 1′,’ FAM-aptamer 2‘ and’FAM-aptamer 3′) or biotin was labeled on terminals of the sequences of the screened three types of aptamers according to a general method so that a binding force may be measured and observed due to a change in polarization of the phosphor according to a binding force to the target.

[0145] 4. Confirmation of Target Binding Force of Screened Aptamer

[0146] After the FAM-aptamer (20 nM) was coated on a 384-well flat-bottom plate, the concentration of a target protein, SFTS virus nuclear protein (NP), was added to the plate at a concentration of 0 to 25 μM and shaken for 5 minutes, and then incubated at room temperature for 30 minutes. Then, the expression intensities at an excitation wavelength of 480 nm and an emission wavelength of 535 nm were measured using a Cytation 5.0 Plate Reader (BioTek) and Gen5 version 2.09.1, and an anisotropy value was plotted against an X-axis with an increasing concentration and then a dissociation constant Kd was measured using a saturation one-site fitting model, and the results were shown in FIGS. 3A to 3C.

[0147] As illustrated in FIGS. 3A to 3C, it was confirmed that the dissociation constant of FAM-aptamer 1 was 3.9 μM, the dissociation constant of FAM-aptamer 2 was 1.8 μM, and the dissociation constant of FAM-aptamer 3 was 0.8 μM.

[0148] From the results, it can be seen that the three types of aptamers screened according to the present invention have the difference above, but all have excellent binding affinity, and in particular, in the case of FAM-aptamer 3, the binding affinity is very excellent. Furthermore, it can be seen from these results that the modified SELEX method according to the present invention is very effective in screening excellent aptamers.

[0149] 5. Confirmation of Antigen Specificity of Screened Aptamers

[0150] A serum containing another type of hantavirus (Hanta) or influenza A virus (InfA) similar in structure to the nuclear protein of SFTS, a serum containing the nuclear protein of SFTS, and a serum (Mix) combined with these serums were added to a 96-well plate coated with a biotin-labeled aptamer-3 (hereinafter, ‘biotin-aptamer 3’), respectively, and added with Streptavidin bound with HRP, and then reacted, and thereafter, color changes were confirmed and the result was illustrated in FIG. 4.

[0151] In addition, the biotin-aptamer 3 was applied to a conventional liposome-based colorimetric sensor platform to determine whether a target was detected, and the result thereof was shown in FIGS. 5A and 5B.

[0152] As illustrated in FIG. 4, it was confirmed that the absorbance was 0.1 a.u. in the serum containing only the Hanta virus or influenza A virus, which had a similar structure to the nuclear protein of SFTS, whereas in the serum containing the nuclear protein of SFTS and the mixed serum, the absorbance was 0.7 a.u.

[0153] In addition, as illustrated in FIGS. 5A and 5B, it was confirmed that as the concentration of the target protein was increased, not only the intensity of the color to be developed was increased, but also the absorbance was also increased in a concentration-dependent manner.

[0154] Through the result, it can be seen that the aptamer according to the present invention has binding specificity capable of specifically binding only to the nuclear protein of SFTS, and as the binding degree varies depending on the concentration of the target protein, the concentration of the target protein present in a desired serum may be measured with high sensitivity.

<Example 2> Immunoassay Using Screened Aptamer

[0155] 1. Screening of Target-Specific Aptamer

[0156] The present inventors commercially purchased influenza A NP, influenza B NP, HIV-1 p24 protein, Ebola NP, and SARS-Cov-2 NP, and screened aptamers having binding specificity to each protein by the method of Example 1, and the sequences of the screened aptamers were shown in Table 2. In Table 2 below, the sequences indicated in bold were sequences bound with primers in the aptamer amplification step, and were prepared by different sequences for each aptamer.

TABLE-US-00003 TABLE 2 SEQ ID Protein NO: Aptamer sequence Influenza SEQ ID 5′ TAG GGA AGA GAA GGA CAT A NP NO: 6 ATG ATG GCG TAC GGG GAT GAG GTG ATC GTA GTG GGT TGA CTA GTA CAT GAC CAC TTG A 3′ Influenza SEQ ID 5′ ATT ATG GCG TTT GCA GCG B NP NO: 7 TTC TGG TTG GTG GTG GTG ATA GGT GGG GGG AAG GAG GGT ATC TTG TTG GTG AGG TAA CGG CT 3′ HIV-I p24 SEQ ID 5′ AGA TAC TGC CAT TCA TTG protein NO: 8 CAT CGA GCA CGC GAC TGA TGA GGA TGG TCT AGT AGC TGG GGT CGA GTA CTA AGC TAT GTG TCG A 3′ Ebola NP SEQ ID 5′ GAT GTG AGT GAC GTG GAT NO: 9 CGA GCG GAT GTG AAG GCT GAA AGT GGC TTT GGG CGG TCG TAA GTG TCA CAG AGC ATG CAA CAA GAC C 3′ SARS-Cov-2 SEQ ID 5′ ATC CAG AGT GAC GCA GCA NP NO: 10 AAC CCA AGC AAA CTA CCT CTA TAC CCT TCG ACC TTC ATC ATG GAC ACG GTG GCT TAG T 3′

[0157] 2. Heterogeneous Sandwich Immunoassay Using Screened Aptamer

[0158] The present inventors examined the detection efficiency of H-sandwich RPA by detecting various concentration ranges of five model target proteins using the selected aptamers (FIG. 6).

[0159] Anti-target antibody (1 ng/mL) in a 0.1 M carbonate buffer (pH 9.6) was immobilized in a 96-well microplate at 4° C. overnight. The coated wells were washed 3 times with PBS-T and blocked with 1% BSA (w/w). Recombinant target proteins and aptamers (1 μM) at various concentrations were pre-incubated at room temperature for 30 minutes. For clinical sample analysis, a binding buffer containing 2% Triton™ X-100 as an assay diluent to lyze the virus was used. The target protein-aptamer mixture was loaded into the coated wells and incubated for 45 minutes at room temperature while gently shaking. An RPA solution containing a primer, dNTP, a reaction buffer and magnesium acetate (MgOAc) was added to the H-sandwich complex containing the antibody-antigen-aptamer to induce isothermal amplification, and an intercalating dye (0.5× EvaGreen®) to facilitate detection of a fluorescence signal was added to the wells and incubated at 37° C. for 20 minutes. Thereafter, the fluorescence signal was measured at an excitation wavelength of 500 nm in an emission wavelength of 530 nm. The fluorescence intensity of the reaction without the target protein was used as a negative control group (Ic), and the fluorescence signal after the RPA reaction with the target protein was recorded as a fluorescence intensity (I). Normalized ratios of fluorescence intensities were calculated using a difference in signal values as follows:

[0160] Relative fluorescence intensity or ΔI=(I−Ic)/Ic.

[0161] 3. Optimization of Heterogeneous Sandwich Immunoassay

[0162] H-sandwich RPA was applied to optimize conditions of factors affecting the detection efficiency to detect a model target (FIGS. 7A to 7D).

[0163] (1) Concentration of Antibody and Aptamer

[0164] First, the detection efficiency according to the concentration of capture antibodies immobilized in the wells and the concentration of the aptamers was examined. The concentrations of the capture antibodies and the aptamers were one of main factors of H-sandwich RPA, and this was because an appropriate concentration of antibodies needs to be immobilized in the wells to capture the antigens and amplify only the antigen-bound aptamers through the RPA reaction.

[0165] As illustrated in FIG. 7A, when the antibody was immobilized at a concentration of 0.1 to 100 ng/mL, and 1 ng/mL of antibody was added while 1 pM of the aptamer and 0.5 pg/mL of influenza A NP reacted with each other, the highest relative fluorescence intensity was shown.

[0166] Next, the concentration conditions of the aptamer reacting with the antigen were examined (FIG. 7B).

[0167] Influenza A NP 0.5 pg/mL was used, and the experiment was performed under aptamer conditions of 0.01 to 10 μM, which was 1 to 1000 times larger than the antigen concentration. The highest relative fluorescence intensity was observed at 1 μM of the aptamer, which was 100 times larger than the antigen concentration.

[0168] Therefore, a subsequent experiment was performed under conditions of 1 ng/mL of the coated antibody and 1 pM of the aptamer.

[0169] (2) Concentration of Intercalating Dye

[0170] In addition, by examining a dilution ratio of an intercalating dye, the concentration of the dye representing the highest relative fluorescence intensity value in the on-off of the antigen was determined. The reacted antigen concentration was 0.5 pg/mL and the aptamer used was 1 pM.

[0171] The intercalating dye was diluted from a 20× stock to 0.05, 0.1, 0.5 and 1×, and the measured fluorescence signals were illustrated In FIG. 7C.

[0172] The fluorescence intensity was increased according to a higher dilution ratio, and when 0.5× intercalating dye was added, the highest relative fluorescence intensity was exhibited.

[0173] Therefore, in an additional experiment, the dilution ratio of the intercalating dye was set to 0.5×.

[0174] (3) Saturation Time of Fluorescence Intensity

[0175] Finally, the present inventors examined the saturation time of the fluorescence intensity generated by the intercalating dye during the RPA reaction.

[0176] As illustrated in FIG. 7D, the fluorescence signal increased rapidly for 8 to 10 minutes, and the reaction time was saturated at 15 minutes. In general, the RPA reaction showed a slight time and a difference in fluorescence signal intensity depending on the enzyme working efficiency, and 20 minutes, which were a recommended condition of an RPA kit, was selected to compensate for these the time and the difference.

[0177] (4) Aptamer Amplification Method

[0178] In addition, the dsDNA detection efficiency of the intercalating dye used in the present invention was confirmed.

[0179] To determine whether the intercalating dye added to the amplification reaction showed increased fluorescence intensity with the dsDNA generated by the amplification process, normal PCR, solution RPA and H-sandwich RPA were performed. The amounts of primers, aptamers and other reagents used in each amplification reaction were the same as each other, normal PCR was performed in 30 cycles, and the RPA reaction time at 37° C. was 20 minutes.

[0180] The primers used for the amplification reaction were shown in bold in Table 2, and specifically, a forward primer was the same as a sequence shown in bold in Table 2 and a reverse primer had a sequence complementary to the sequence shown in bold in Table 2.

[0181] As illustrated in FIGS. 8A and 8B, a difference in fluorescence signal before and after amplification occurred in each method, and the relative fluorescence intensity was highest in the solution RPA, but was not significantly different from that of the H-sandwich RPA. In each case, the amplified DNA was confirmed through gel electrophoresis, and a band difference according to the presence or absence of the antigen was clearly observed in the H-sandwich RPA (FIG. 8B).

[0182] In the normal PCR, the high amplification reaction temperature may affect the detection efficiency of the intercalating dye, and since the H-sandwich RPA amplifies the aptamers on the immune complex bound to the wells, it is supposed that the on-off difference on the gel is obvious.

[0183] 4. Effects of Immunoassay Under Optimal Conditions

[0184] In order to examine the effects of the immunoassay of the present invention to which the optimized conditions were applied, fluorescence signals after RPA reaction were measured using model targets and target-specific aptamers in various concentration ranges.

[0185] After the capture antibody of each target was immobilized in the well, premixed antigens and aptamers were loaded into the wells and incubated to form immune complexes. After the heterogeneous sandwich immune complex (antibody-antigen-aptamer) was constructed, unbound aptamers were removed through a sufficient washing step. Thereafter, the primers, the RPA reagents and the intercalating dye were added to the wells and incubated at 37° C. for 20 minutes to induce the RPA reaction and fluorescence signals generated from the amplified aptamers were measured.

[0186] As illustrated in FIGS. 9A to 9E, the fluorescence signal of the influenza A virus NP was detected at a concentration of 1 fg/mL, which was confirmed by observing a band corresponding to the aptamer length on an agarose gel.

[0187] In the same procedure as described above, the present inventors also applied the detection method of the present invention to the detection of other virus-related antigens including influenza B virus NP, HIV-1 virus p24, Ebola virus NP, SARS-Cov-2 NP, respectively.

[0188] In addition to the fluorescence intensity measurement, gel bands were observed in the concentration range of 1 and 10 fg/mL (not illustrated), and the relative fluorescence intensity value of the detection limit concentration was 0.5 or less (FIGS. 9B to 9E). As a result of comparing the relative fluorescence intensity of each target with a gel electrophoresis image, no bands were observed in a control group (no target antigen) with a fluorescence signal of less than 0.5 and concentration groups.

[0189] 5. Crossover Possibility Depending on Interfering Substrate

[0190] Next, the crossover possibility of the H-sandwich RPA platform according to the presence of an interfering substance was examined.

[0191] The relative fluorescence intensity was measured in a mixture of influenza A, B virus NPs or p24, Ebola virus NP and human fluid according to the use of each target-specific aptamer (FIGS. 10A and 10B). The relative fluorescence intensity was calculated based on the fluorescence signal detected after performing an experiment by the above-mentioned method in each well divided into groups containing 10-fold diluted human nasal fluid, target or non-target, and target and non-target.

[0192] The relative fluorescence intensity of each group was corrected using the fluorescence intensity of the control well (Ic). In the case of using an aptamer specific to influenza A NP, the target was influenza A NP and the non-target was influenza B NP (blue bar). The target and the non-target were vice versa when an influenza B NP-specific aptamer (pink bar) was used.

[0193] As illustrated in FIG. 10A, THE high relative fluorescence intensity was observed in the presence of the target despite the 10-fold higher non-target concentration, and there was little matrix effect in the diluted nasal fluid containing non-specific DNA and protein. When p24 and Ebola NP-specific aptamers were used as detection probes in 10-fold diluted serum, target, non-target and mixture, respectively, high relative fluorescence intensities were also shown in the presence of the target (FIG. 10B).

[0194] These results prove that the H-sandwich RPA has high selectivity and sensitivity due to its high signal-to-noise ratio. The immunoassay method of the present invention may maximize selectivity by using a target-specific capture antibody and an aptamer, and amplifying only the DNA aptamer in the formed heterogeneous sandwich immune complex. These properties of the proposed method were distinguished from those of conventional immunoassays with low signal-to-noise ratios using the same two bioreceptors.

[0195] 6. Comparison with Various Immunoassay Methods

[0196] The present inventors compared the H-sandwich RPA of the present invention with various immunoassay methods.

TABLE-US-00004 TABLE 1 Multiple Detection method Target LOD detection PCR using short Thrombin 2 pM Impossible DNA aptamers Sandwich ELISA Protective 4.1 ng/mL Possible using DNA antigen encapsulated liposomes Photoelectrochemical HIV-1 p24 10 ng/mL Impossible immunoassay using DNA labeling H-sandwich RPA Virus-related to 1 fg/mL Possible using DNA aptamers protein

[0197] Referring to Table 1, the immunoassay method of the H-sandwich RPA enables multiple detection and showed the lowest limit of detection (LOD) compared to other immunoassays. Therefore, the immunoassay method of the present invention has an advantage of detecting the target substance present at a low concentration of 1 fg/mL by selectively amplifying only the aptamer bound to the target substance in the heterogeneous sandwich structure to detect the relative fluorescence signal.

[0198] <Conclusion>

[0199] The present inventors have developed a highly sensitive immunoassay method for the detection of protein biomarkers based on the integration of RPA and immunoassay. The present inventors also newly selected DNA aptamers specific to HIV-1 p24, Ebola virus NP and SARS-Cov-2 NP by the aptamer screening method of the present invention.

[0200] Unlike immuno-PCR using capture and detection antibody pairs, since the H-sandwich RPA uses a DNA aptamer that binds to the antibody-antigen complex in an H-sandwich form, antibody pair screening is not required. The H-sandwich RPA has an advantage of high sensitivity and specificity due to the rapid amplification of target-bound aptamers through the RPA reaction. Through the application of the proposed immunoassay method and optimization of various reagents, a low limit of detection for H-sandwich RPAwas observed with the detection of an attomolar concentration level of a model target protein. This method showed higher detection efficiency for influenza-infected patient samples than commercial ELISA and LFA kits. The approach of the present inventors enables high-sensitivity detection of protein biomarkers in a well-plate, unlike conventional PCR-based amplification methods that may be applied only to nucleic acids. Therefore, the results of the present inventors suggest that the H-sandwich RPA may be universally applied to various targets through appropriate selection of biomarker-specific aptamers.

[0201] In the prior art, Fischer, Nicholas O et al., there is disclosed a technique of immobilizing an anti-protein antibody or a biotinylated protein target to magnetic beads, binding to the aptamer and the amplifying the bound aptamer through PCR. However, in the prior art, the aptamer is separated using magnetic beads and transferred to a tube to perform the amplification reaction. Unlike this, the present invention may be differentiated from the prior art in that aptamer binding, amplification, and detection are enabled on one plate, and multiplex PCR-based detection is enabled by using a target-specific aptamer.